In Vitro Effects of Propranolol on T Helper Type 1 Cytokine Profile in Human Leukemic T Cells

Introduction: Cytokines are a large group of proteins play a key role in inflammation. Down-regulation of pro-inflammatory cytokines has beneficial effect on heart function. Propranolol, as a non selective beta-adrenergic blocker, has been extensively used for treatment of many cardiovascular problems such as arrhythmias and heart malfunction. In addition anti-inflammatory effects of propranolol have been revealed. In this study the propranolol effect on T helper type 1 cytokine profile in human leukemic T cells has been assessed in vitro. Materials and methods: Human leukemic T cells (Molt-4 and Jurkat) were cultured in complete RPMI medium. The cells were then incubated with different concentrations of propranolol (0.03- 30 µM) in the presence or absence of PHA (10 µg/ml) for 48 hours. The supernatants of cell culture media were collected and used for cytokines assay. Results: Propranolol significantly decreased the T helper type 1 cytokine profile [Interleukin-2 (IL-2) and Interferon- γ (IFN-γ)] production in PHA stimulated Molt-4 and Jurkat cells, after 48 hour of incubation time, dose-dependently compared to untreated control cells. Conclusion: Our data showed a dose dependent inhibitory effect of propranolol on the IL-2 and IFN-γ production in human leukemic Molt-4 and Jurkat cells. The anti- inflammatory effect of propranolol reported by other investigators may be in part due to its suppressive effect on production of inflammatory cytokines such as IL-2 and IFN-γ. So, propranolol along with its chronic long-term usage in cardiovascular problems may have potential implication in treatment of inflammatory-based disorders.


INTRODUCTION
Cytokines are a large group of proteins which are produced by a number of cells and play a key role in the immunopathogenesis of several disorders such as inflammatory bowel disease, periodontitis, Sjögren's syndrome and atherosclerosis. [1][2][3][4] In addition deysregulation of cytokines in some patients has been shown. [5][6][7] Modulation of cytokines has had promising results for treatment of some diseases such as leukemia and cardiac infarction. 8,9 Improved heart function following down-regulation of proinflammatory cytokines has been reported. 10,11 Propranolol, as a non selective beta-adrenergic blocker, has been extensively used for treatment of many cardiovascular problems such as ischemic heart diseases, arrhythmias and heart malfunction. 12,13 In addition anti-inflammatory and antiangiogenesis effects of propranolol have been revealed. 14 inflammatory cell and some cytokine (tumor necrosis factor-α and interleukin-8) levels in alveolar fluid of rat by propranolol has been reported in vivo. 16 Besides the regulatory effect of propranolol on cytokine production in tumorinfiltrating lymphocytes and peripheral blood mononuclear cells (PBMCs) of colorectal cancer patients has been shown. 17 In this study the propranolol effect on profile of T helper type 1 (Th1) cytokines in human T cell lines has been assessed in vitro.

Preparation of propranolol
Propranolol was dissolved in RPMI-1640 medium and stored at -20°C until use. Drug was diluted in culture medium to prepare the needed concentrations before use.

Cell culture and treatment
The method has been described in detail elsewhere. 15 Briefly, the human leukemic cells were cultured in RPMI-1640 medium supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (100 µg/ml) at 37°C in 5% CO 2 . The cells were seeded at a density of 2×10 6 cell/ml and then incubated with different concentrations of propranolol (0.03-30 µM) in the presence or absence of PHA (10 µg/ml) for 48 hours. The supernatants of cell culture media were collected and used for IL-2 and IFN-γ assay. All experiments were done in triplicate.

Cytokines assay
The amount of IL-2 and IFN-γ secreted in the cell culture supernatants by human leukemic T cell lines (Molt-4 and Jurkat) was measured with the Quantikine human enzyme-linked immunosorbent assay (ELISA) kits (R & D systems) according to the manufacturer's instructions. This assay uses the quantitative sandwich enzyme immunoassay technique. Complete RPMI medium was used as control and human recombinant IL-2, and IFN-γ were employed as standard for drawing the standard curves.

Statistical analysis
Effect of the drug on each cell line was performed in three independent experiments and the results were expressed as mean ± SEM. Statistical comparisons among groups were made by analysis of variance (ANOVA). P<0.05 was considered significant. Test of multiple comparison of Tukey was applied (5%) for statistically significant differences. For statistical analysis and graph making, the software SPSS 11.5 and Excel 2013 were used, respectively.  14 may be in part due to its suppressive effect on IFN-γ production. Also, in our study propranolol significantly reduced IL-2 production in human leukemic T cells. As IL-2 has an imperative role in inflammation 19 the antiinflammatory effect of propranolol reported by other investigators 14 may be partially owing to its inhibitory effect on IL-2 production. Reduction of IL-2 by propranolol showed in our study is consistent with Ebbinghaus et al. study 20 in which propranolol considerably reduced IL-2, IL-17 and transforming growth factor B in supernates of murine lymph nodes and spleen lymphocytes. Moreover our findings are in accordance with Zhou et al. study in which propranolol decreased inflammatory cell and cytokines (tumor necrosis factor-α and interleukin-8) levels in alveolar fluid of rat. 16 In addition decrease of proinflammatory cytokines such as IL-1β and TNF-α in an experimental model of periodontal disease by propranolol have been shown. 21 Besides pretreatment with propranolol reversed increase of IL-6 and TNF-α in a mice model. 22 According to our previous study, 23   Decrease of inflammatory cytokines (IFN-γ, IL-2) by propranolol in the present study is in accordance with the decrease of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) by propranolol in our previous study 15 because VEGF and MMP-2 have important roles in inflammation. 24,25 Implication of anti-inflammatory mediators in treatment of ischemic heart failure has been reported. 26,27 So, positive effect of propranolol on treatment of many cardiovascular problems such as ischemic heart diseases and heart malfunction 12,13 may be in part owing to its anti-inflammatory effects through inhibition of inflammatory cytokines (IL-2 and IFN-γ) was shown in this study.

I)Propranolol effect on IL-2 production in human leukemic cell lines Propranolol effect on IL-2 production in human
It should be considered that in our study, the concentration of propranolol which decreased the IL-2 and IFN-γ production after 48 hours incubation time in vitro was higher than that of usually used in cardiovascular patients. Of course it should be noted that our study was performed in vitro which is different situation from in vivo in patients. Furthermore, as mentioned before, in Seyedi et al. study 17 propranolol decreased the IFN-γ and IL-17 production at much lower concentration (1 µM) but higher incubation time (72 hours) than us. As propranolol is used in cardiovascular diseases for long time period, 28 the drug concentration used in patients might be enough for inhibiting of inflammatory cytokines such as IL-2 and IFN-γ in vivo. In our study, propranolol reduced IFN-γ and IL-2 (Th1 cytokines) production. As Th1 cells have essential anti-tumor properties, 29 we might conclude that propranolol decreases anti-cancer immune responses. But antiproliferative and anticancer effects of propranolol have been reported by other researchers. 30,31 So it seems that anti-tumor effects of propranolol may be mediated by Th1independent mechanisms. This deduction is consistent with our previous study 15 in which propranolol reduced VEGF and MMP-2 production, since VEGF and MMP-2 have important roles in tumor progression and metastasis. 32,33 Also the important role of some pro-inflammatory cytokines in tumorigenesis has been shown. 34 Thus the antitumor effects of propranolol in some cancers pronounced by other investigators may be in part due to its inhibitory effects on certain proinflammatory cytokines. According to our results propranolol could be considered as a potential inhibitor of proinflammatory cytokines and so may have beneficial effects for treatment of inflammatory-based disorders including allergic and autoimmune diseases.
Altogether propranolol as well as its long-lasting use in cardiovascular disorders might be an effective anti-inflammatory agent and have potential implication in management of inflammatory conditions. Further studies about propranolol effects on other inflammatory cytokines/ markers in vitro as well as inflammatory-based diseases in vivo are also needed.

CONCLUSION
Propranolol can be considered as a potential inhibitor of pro-inflammatory cytokines and along with its chronic long-term usage in cardiovascular problems could have potential implication in management of inflammatory-based disorders.

ACKNOWLEDGEMENT
Thanks are due to Dr. Razavi for scientific consultations.

CONFLICT OF INTEREST
There is no conflict of interest related to this article.